The long term objectives of this proposal is to understand how the initiation step of DNA replication is regulated. Previous studies investigated the molecular mechanisms underlying gene amplification, a type chromosomal rearrangement that does not occur in normal somatic cells, but occurs in many clinically important human cancers. A "bubble- breakage" model fore gene amplification evolved from these studies; it postulates that entry into 5-phase under metabolically limiting conditions precipitates the formation of amplicons by chromosome breakage within slowed or stalled replication bubbles. The amplicons are proposed to be autonomously replicating large chromosome fragments or small fragments such as double minute chromosomes (DMs) or their episomal precursors. This work further emphasized the need to identify the regions within which DNA replication initiates, and to develop functional assays to enable their molecular dissection. The first two Specific Aims propose to identify, isolate, and characterize the cis acting sequences that enable bidirectional DNA replication to initiate. Three putative initiation regions were identified and cloned from episomes during the past grant period. They do not enable autonomous replication of transfected sequences, but they do function in the genome. This raises the possibility that origin function might depend on an "imprinting" process occurring within a chromosome. A biochemical assay for origin activity, and a molecular strategy employing FLP, a yeast site specific recombinase adapted to function in mammalian cells, will be used to test this idea. Furthermore, one initiation region appears to contain a replication fork barrier which will be analyzed using biochemical and genetic strategies. The third specific aim proposes to investigate structure-function relationships in replication origins in their native chromosomal location. This is an essential goal to achieve, since one must ultimately have a means of investigating how sequences in the vicinity of the origin impact on origin activity. The strategy employs homologous recombination to produce specific alterations in regions shown in the first two specific aims to be important for origin function. These studies will be performed in cell lines that have been made hemizygous for the candidate origin region using one of several new methods for deleting specific regions of the mammalian genome. One method involves a combination of homologous and FLP-mediated recombination to generate targeted deletions of large genomic regions and to produce an autonomous episome harboring specific origins as a reciprocal The multifaceted approach to the analysis of replication origins within and outside of the chromosome should elucidate the minimal regions required for origin function, and enable a mob precise molecular understanding of those features that contribute to origin function and the initiation of DNA replication.